The present invention relates to a novel microorganism Streptomyces megasporus SD5. The present invention also relates to a process for the isolation of said Streptomyces megasporus SD5. The invention also relates to a novel fibrinolytic enzyme actinokinase extracted from said microorganism and to a process for the extraction of said enzyme. In another aspect, the invention also pertains to a method for the treatment of thrombolytic disorders using said enzyme.
Thrombosis of critical blood vessels results in the loss of blood flow to vital organs thereby resulting in mortality. In vivo formation of thrombus is a pathological consequence of interactions between the hemostatic mechanism of blood and the components of injured vessels. [Collen, On the future of thrombolytic therapy in acute myocardial infarction, Haber E., and Braunwald E., editors Thrombolysis: Basic contribution and clinical progress, 1991, St. Louis, Mo., Mosby, p. 131]. In recent years, the treatment of thrombosis has focused on pharmacological dissolution of the thrombus. Thrombolytic therapy has now become the established procedure for recanalizing the occluded myocardial blood vessels and thereby reducing mortality. Thrombolytic agents are plasminogen activators that convert plasminogen, the inactive proenzyme of the fibrinolytic system of blood to the active proteolytic enzyme, plasmin. Several plasminogen activators are disclosed in the prior art such as streptokinase [Castellino, et al, 1993, Met.in.Enz, 222, 244-255]; staphylokinase [Trien, et al, 1993, Met.in.Enz, 223, 156-167]; urokinase [Barlow, 1993, Met.in.Enz, 222, 239-243] and tissue plasminogen activators [Lijnen H. R. and Collen D., 1993, Met.in.Enz, 223, 197-205], both in native and recombinant forms.
The search for therapeutic agents useful in the treatment of thrombosis has focused on fibrinolytic enzymes. In the search for new enzymes, attention is being given to living sources such as bacteria in view of the potential for greater efficacy [Kim H. K., Kim, G. T., Kim, D. K., Won, A. C. Park, S. H., Jeong, Y. K. and Kong, I. S. 1997. Purification and characterization of a novel fibrinolytic enzyme from Bacillus sp. KA 38 originated from fermented fish. J. Ferment. Bioengineering 84 (4), 307-312; Wang, J., Wang, M. and Wang Y 1999. Purification and characterization of a novel fibrinolytic enzyme from Streptomyces sp. Clin. J. Biotech. 15 (2) 83-89]. However, these enzymes are very expensive [Thomson, P. L., Tonkin, A. M., Aylward, P and White, H. 1993, Thrombolysis ""93 Overview and conclusions. Australian New Zealand Journal of Medicine 23: 774-777] and occasionally show haemorrhagic side effects [Collen, D., Lijnen, H. R. and Stassen, J. N. 1993. Combination of animal models and biotechnology in the development of new thrombolytic agent, Fibrinolysis, 7, 36-43]. As a result it is necessary to develop new fibrinolytic enzymes that are less expensive and effective without any adverse effects.
Of the enzymes known in the art, streptokinase has predominantly been used for coronary superfussion. However, streptokinase is expensive and the origin is a pathogen. Another disadvantage in the use of streptokinase as a thromobolytic agent is that the failure rate is reportedly as high as 25%. As has been explained, known thrombolytic agents have limited efficacy and potentially life threatening side effects. Other disadvantages of known agents include requirement of large therapeutic doses, limited fibrin specificity, short half life, re-occlusion and bleeding complications.
The limitations of known thrombolytic agents can also be explained on the basis of the heterogeneity of coronary arterial thrombus, which consists of both etythrocyte and plate rich zones and the knowledge of the mechanism of fibrin dissolution and platelet de-aggregation. Prior art focuses on dissolution of whole blood clot. For example, streptolkinase and urokinase do not show any affinity for fibrin and activate circulating and fibrin bound plasminogen indiscriminately. As a result, initially formed plasmin is neutralized by xcex10-antiplasmin resulting in reduction or no action on thrombolysis, The potential of pharmacological dispersion of platelet clumps and platelet rich thrombus has not been fully explored as yet. Also, attention has not been given to complementary treatment of both the whole blood clot and the platelet clumps and platelet rich thrombus.
In view of the importance of fibrinolytic enzymes in the treatment of thrombosis in human beings, microbial sources including Streptomyces are being investigated to obtain compounds that could activate any one site in the complex and diverse fibrinolytic pathway. Interest in the search of novel thermophilic Streptomyces as producers of bioactive but non-antibiotic metabolites has increased because of thermophilic Streptomyces strains, associated with the tectonically active zones, are considered a unique and important genetic resource due to the intrinsic stability of their bioactive molecules which are extremely useful for large scale production under harsh conditions, in terms of pH and temperature.
The main object of the present invention is to develop novel thrombolytic enzymes from natural microbial sources, which are inexpensive and show increased efficacy.
It is another object of the invention is to obtain new strains of microbial organisms that could provide compounds for use as inexpensive and efficacious thrombolytic agents.
It is a further object of the invention to provide a process for the growth of new strains of microbial organisms, which could provide a source for novel compounds for use as inexpensive and efficacious thrombolytic agents.
It is another object of the invention to provide a process for the extraction of novel enzymes from isolated Streptomyces organisms, which are useful as thrombolytic agents by dispersion of platelet clumps and platelet rich thrombus.
It is another object of the invention to provide a method of treatment of thrombosis using a novel fibrinolytic enzyme obtained from a new strain of Streptomyces bacteria, which has greater efficacy as compared to the substances employed in the art.
Recent interest in biotechnology and biodiversity has prompted the search of novel thermophilic Streptomyces, producers of bioactive but nonantibiotic metabolites. Thermophilic Streptomyces strains associated with tectonically active zones are considered unique and important genetic resource due to the intrinsic stability of their bioactive molecules which are extremely useful for large scale production under harsh conditions, in terms of pH and temperature. Tectonically active zones prevail along the Western coast of Maharashtra, India. There are 60 thermal springs with a temperature range of 40xc2x0 C.-90xc2x0 C. in this region. Another unique site is the Lonar lake basin, a meteoritic crater, situated on the Deccan plateau. These ecosystems were selected for isolation sites as these are tectonically active, confined but microbiologically untouched area and their physicochemical properties are relatively constant. The present invention concerns a novel strain of Streptomyces (named Streptomyces megasporus SD5 by the applicants) isolated from such thermal springs.
Accordingly the present invention relates to a novel strain of Streptomyces named Streptomyces megasporus SD5 and variants and mutants thereof.
In one embodiment of the invention, Streptomyces megasporus SD5 comprises elongate colourless branched substrate mycelia and short aerial mycelia.
In another embodiment of the invention, the microorganism Streptomyces megasporus SD5 hydrolyses casein and starch and does not liquefy gelatine in different media, form H2S or reduce nitrate.
In another embodiment of the invention, the microorganism Streptomyces megasporus SD5 does not produce melanoid pigment on tyrosine sugar, peptone-yeast extract, iron agar and tryptone yeast extract broth.
In another embodiment of the invention, the microorganism Streptomyces megasporus SD5 has the following carbon assimilation pattern in Pridham-Gottileb medium:
Positive: casein, starch, glucose and fibrin
Weak: cellulose and gelatine
Negative: tributyrin, pullulan, xylan and cyclodextrin
In another embodiment of the invention, the microorganism Streptomyces megasporus SD5 is inhibited by thiostrepton, and lincomycin at a concentration of 50 xcexcg mlxe2x88x921, tolerates NaCl concentration up to 1% and has a pH tolerance of from 6.0 to 9. 0.
In another embodiment of the invention, the microorganism Streptomyces megasporus SD5 has the profile given below in Table I.
The present invention also relates to a process for the isolation of the microorganism Streptomyces megasporus SD5 from hot water sources and springs using a nutrient medium containing carbon and nitrogen sources in a definite ratio and inorganic nutrient salts.
In one embodiment of the invention, the isolation of the microorganism Streptomyces megasporus SD5 is done using a solidifying agent in combination with the nutrient medium.
In one embodiment of the invention, the carbon sources are selected from the group consisting of glucose, starch, sucrose and agricultural wastes.
In another embodiment of the invention, the nitrogen sources are selected from the group consisting of peptone, yeast extract, beef extract, malt extract, casein hydrolysates, haemoglobin and fibrin.
In another embodiment of the invention, the solidifying agents are selected from agar and agarose.
In yet another embodiment of the invention, the inorganic salts are selected from salts of calcium, sodium, potassium and magnesium.
In another embodiment of the invention, the nutrient medium comprises glucose in an amount of 0.2 to 1 g, yeast extract in an amount of 0.1 to 0.6 g, glycerol in an amount of 0.2 to 0.5 g, NaCl in an amount of 0.1 to 0.6 g and Agar as the solidifying agent.
In a further embodiment of the invention, the nutrient medium comprises glucose in an amount of 0.5 g, yeast extract in an amount of 0.5 g, glycerol in an amount of 0.5 g, NaCl in an amount of 0.5 g and Agar in an amount of 2 g as the solidifying agent.
In another embodiment of the invention, the nutrient medium comprises glucose in an amount of 1.0 g, L-asparagine in an amount of 0.5 g, yeast extract 0.5 g, peptone in an amount of 0.5 g and agar as the solidifying agent.
In another embodiment of the invention, the nutrient medium comprises (% w/v) glucose 1, yeast extract 0.5, peptone 0.5, NaCl 0.5 and CaCl2 0.2 with the pH being 8.0.
In a further embodiment of the invention, the nutrient medium comprises starch in an amount of 2.0 g, casein in an amount of 1 g and NaCl in an amount of 0.5 g.
In another embodiment of the invention, the microorganism Streptomyces megasporus SD5 has the following profile as given in Table I below:
In another embodiment of the invention, the microorganism Streptomyces megasporus SD5 is mutated using conventional mutagens selected from the group consisting of N-methyl-Nxe2x80x2-nitro-N-nitrosoguanidine, ethyl methyl sulfonate and exposure to UV radiation.
The present invention also relates to a novel enzyme named actinokinase extracted from microorganism Streptomyces megasporus SD5, which is useful as a thrombolytic agent.
The present invention also relates to a process for the preparation of enzyme actinokinase which comprises cultivating the microorganism Streptomyces megasporus SD5 or a variant or mutant thereof under aerobic conditions at a temperature in the range of 45xc2x0 C. to 65xc2x0 C. in an alkaline aqueous nutrient medium and recovering the enzyme.
In one embodiment of the invention, the nutrient medium comprises of carbon sources, nitrogen sources, mineral salts and trace elements and is at a pH of 7.5 to 9.0.
In another embodiment of the invention, the carbon source is selected from the group consisting of glucose, soluble starch and raw starch.
In another embodiment of the invention, the nitrogen source is selected from the group consisting of yeast extract, peptone, tryptone, casein and casein hydrolysate.
In a further embodiment of the invention, the inorganic salts used are selected from the group consisting of chlorides of calcium, sodium, potassium and magnesium.
In another embodiment of the invention, the trace elements are selected from the group consisting of iron, manganese, copper, zinc, borate and molybdenum.
In another embodiment of the invention, the temperature is maintained in the range of 50xc2x0 C.-55xc2x0 C. and the cultivation is carried out by fermentation, preferably submerged fermentation.
In another embodiment of the invention, an anti-foam agent, preferably soybean oil is added to the nutrient medium during fermentation.
In yet another embodiment of the invention, the enzyme is concentrated with ammonium sulphate and then separated from other proteins.
In a further embodiment of the invention, the concentrated enzyme is separated by gel filtration, ion exchange chromatography and isoelectric focusing in the presence of a buffer having a pH in the range of 3-10.